Truncated epiderimal growth factor receptor (EGFRt) for transduced T cell selection

ABSTRACT

A non-immunogenic selection epitope may be generated by removing certain amino acid sequences of the protein. For example, a gene encoding a truncated human epidermal growth factor receptor polypeptide (EGFRt) that lacks the membrane distal EGF-binding domain and the cytoplasmic signaling tail, but retains an extracellular epitope recognized by an anti-EGFR antibody is provided. Cells may be genetically modified to express EGFRt and then purified without the immunoactivity that would accompany the use of full-length EGFR immunoactivity. Through flow cytometric analysis, EGFRt was successfully utilized as an in vivo tracking marker for genetically modified human T cell engraftment in mice. Furthermore, EGFRt was demonstrated to have cellular depletion potential through cetuximab mediated antibody dependent cellular cytotoxicity (ADCC) pathways. Thus, EGFRt may be used as a non-immunogenic selection tool, tracking marker, a depletion tool or a suicide gene for genetically modified cells having therapeutic potential.

PRIORITY CLAIM

This application is a divisional of U.S. application Ser. No.14/340,512, filed Jul. 24, 2014, which is a divisional of U.S.application Ser. No. 13/463,247, filed May 3, 2012, issued as U.S. Pat.No. 8,802,374, which is a continuation of International Application No.PCT/US2010/055329, filed Nov. 3, 2010, which claims the benefit of U.S.Provisional Application No. 61/257,567, filed Nov. 3, 2009, the subjectmatter of which is incorporated by reference as if fully set forthherein.

TECHNICAL FIELD

The present products and methods relate to the fields of immunology andpurification of genetically modified cells, specifically to a truncatedor otherwise modified receptor paired with a corresponding antibody,such as a polypeptide derived from human epidermal growth factorreceptor (EGFR) paired with cetuximab, for use in cancer immunotherapy.

BACKGROUND

Immune cell products with homogenous expression of tumor targetingchimeric antigen receptors (CARs) are desirable for clinical evaluationof adoptive therapy strategies to eliminate the product-to-productvariability of transgene expression otherwise intrinsic to transductionand other genetic modification procedures without subsequent selection.Immunotherapy using genetically redirected immune cells is an attractiveapproach for treating minimal residual disease in a variety of cancerpatients. However, immunologic rejection of cell products expressingantibiotic selection proteins as part of the transduction strategy hasimpeded this strategy. A novel selection marker that is not expressed onhuman lymphocytes, does not contain endogenous signaling or traffickingfunction, and is recognized by a known, preferably commerciallyavailable, pharmaceutical grade antibody reagent that can be utilizedfor selection, in vivo tracking, and depletion of transduced cells wouldbe a significant improvement in the art.

SUMMARY

Products and methods for purification, both in vivo and ex vivo, ofgenetically modified cells are provided herein. The genetically modifiedcells may be modified by transduction, or any other process that adds,deletes, alters, or disrupts an endogenous nucleotide sequence. Thegenetically modified cells may be transduced T cells with alteredactivity, including altered immunoactivity.

According to the embodiments described herein, a non-immunogenicselection epitope compatible with immunomagnetic selection facilitatesimmunotherapy in cancer patients without undesirable immunologicrejection of cell products (i.e. as seen when expressing antibioticselection proteins) may be generated by removing certain amino acidsequences of the protein. In some embodiments, the non-immunogenicselection epitope is a gene encoding an endogenous cell-surface moleculethat is modified or truncated to retain an extracellular epitoperecognized by a known antibody or functional fragment thereof, and toremove any signaling or trafficking domains and/or any extracellulardomains unrecognized by the known antibody. The removal of the signalingor trafficking domains and/or any extracellular domains unrecognized bythe known antibody renders the endogenous cell-surface molecule inert,which is a desired property for the molecule. The non-immunogenicselection epitope may also be used for as a selection tool or trackingmarker.

The modified endogenous cell-surface molecule may be, but is not limitedto, any cell-surface related receptor, ligand, glycoprotein, celladhesion molecule, antigen, integrin or cluster of differentiation (CD)that is modified as described herein. In some embodiments, the modifiedendogenous cell-surface molecule is a truncated tyrosine kinasereceptor. In one aspect, the truncated tyrosine kinase receptor is amember of the epidermal growth factor receptor family (e.g., ErbB1,ErbB2, ErbB3, ErbB4).

Epidermal growth factor receptor, also known as EGFR, ErbB1 and HER1, isa cell-surface receptor for members of the epidermal growth factorfamily of extracellular ligands. Alterations in EGFR activity have beenimplicated in certain cancers. In a first aspect, a gene encoding anEGFR polypeptide comprising human epidermal growth factor receptor(EGFR) that is constructed by removal of nucleic acid sequences thatencode polypeptides including the membrane distal EGF-binding domain andthe cytoplasmic signaling tail (a “truncated EGFR” or “EGFRt”), butretains the extracellular membrane proximal epitope recognized by ananti-EGFR antibody. Preferably, the antibody is a known, commerciallyavailable anti-EGFR monoclonal antibody, such as cetuximab, matuzumab,necitumumab or panitumumab.

Application of biotinylated-cetuximab to immunomagnetic selection incombination with anti-biotin microbeads successfully enriches T cellsthat have been lentivirally transduced with EGFRt-containing constructsfrom as low as 2% of the population to greater than 90% purity withoutobservable toxicity to the cell preparation. Constitutive expression ofthis inert EGFRt molecule does not affect T cell phenotype or effectorfunction as directed by the coordinately expressed chimeric antigenreceptor (CAR), CD19R. Through flow cytometric analysis, EGFRt wassuccessfully utilized as an in vivo tracking marker for T cellengraftment in mice. Furthermore, EGFRt was demonstrated to have suicidegene potential through Erbitux® mediated antibody dependent cellularcytotoxicity (ADCC) pathways. Thus, EGFRt may be used as anon-immunogenic selection tool, tracking marker, and suicide gene fortransduced T cells that have immunotherapeutic potential. The EGFRtnucleic acid may also be detected by means well known in the art.

In another embodiment, methods of discovering and designing modified,truncated or altered endogenous cell-surface molecules which bind toantibodies, preferably commercially available antibodies, as describedherein are provided. The methods include modeling the protein ofinterest and truncating functional portions, while leaving theantibody-binding portions intact. The resulting modified receptor orligand can be sorted using a labeled antibody and then enriched suchthat the concentration of the modified receptor or ligand is increased.

Yet another embodiment provides a method of selecting transduced T cellscomprising transducing T cells with a modified, truncated or alteredendogenous cell-surface molecule gene sequence (e.g., truncated EGFR)and then applying an antibody that binds the modified ligand or receptorsequence to the transduced T cells. If the modified receptor sequence isEGFRt, the antibody is preferably a biotinylated anti-EGFR monoclonalantibody. The T cells are then sorted by adding anti-biotin microbeadsand selecting the T cells using immunomagnetic separation, addingfluorochrome-conjugated anti-biotin and selecting the T cells usingFluorescence Activated Cell Sorting, or any other reliable method ofsorting the cells. The modified ligand or receptor sequences, such asthe EGFRt sequence, may be contained in a suitable transfer vehicle suchas a lentiviral vector.

These and other embodiments are further explained in the drawing anddetailed description herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a molecular model of EGFR vs. EGFRt proteins based on thecrystal structure files. The EGFR structure on the left shows afull-length EGFR with the structure of the four extracellular domains(Domains I-IV). The middle structure shows the truncated EGFR (EGFRt),which is missing Domain I, Domain II, the Juxatmembrane Domain, and theTyrosine Kinase Domain as compared to an unmodified EGFR. The EGFRt onthe right shows truncated structure bound to Eribitux® Fab, comprised ofV_(H)-C_(H1) and V_(L)-C_(L). The domains are separated with dottedlines.

FIGS. 2a-d illustrate the selection of EGFRt⁺ T cells using biotinylatedcetuximab (referred to in the figure as Erbitux®). FIG. 2a is aschematic of the cetuximab biotinylation and reformulation process. FIG.2b is a graph showing titration of biotinylated cetuximab. 10⁶ EGFR⁺cells were stained with either 0 μg (black), 1.45 μg (red), 0.145 μg(orange), 14.5 ng (yellow), 1.45 ng (green), 0.145 ng (blue) or 14.5 μg(purple) of biotinylated cetuximab followed by 0.5 □μg PE-conjugatedstreptavidin and analyzed by flow cytometry. 14.5 ng or more ofbiotinylated cetuximab was deemed sufficient for future staining. FIG.2c depicts schematics of both the immunomagnetic (top) and thefluorescence activated cell sorting (bottom) EGFRt selection procedures.

FIG. 2d shows immunomagnetic selection of various T cell lineslentivirally transduced with CAR and EGFRt containing constructs.Schematics of the CD19CAR-T2A-EGFRt (left) andCD19CAR-T2A-EGFRt-IMPDH2dm (right) constructs contained in lentiviralvectors are shown above the corresponding pre- and post-selection flowcytometric analyses for surface EGFRt expression. Codon optimizedsequence portions of the CD19-specific, CD28 co-stimulatory CAR,followed by the self-cleavable T2A, EGFRt and IMPDH2dm selection markersare indicated, along with the Elongation Factor 1 promoter sequences(EF-1p), and the GCSFR alpha chain signal sequences (GCSFRss, whichdirects surface expression). Flow cytometric analysis of lentivirallytransduced T cell lines that had been stained with abiotinylated-cetuximab antibody and PE-conjugated anti-biotin antibody(black histograms) was performed on both the input T cells (PRE SLXN)and the positive fraction obtained from AutoMACS™ (POS FRXN). Openhistograms represent staining with PE-conjugated anti-biotin antibodyalone, and the percent positive cells are indicated in each histogram.Selection of CD19CAR⁺EGFRt⁺ Line A occurred 3 days after transduction ofT cell blasts. Selection of CD19CAR⁺EGFRt⁺ Line B occurred after 3 REMstimulations of transduced CMVpp65-specific T_(CM)-derived cells.Selection of CD19CAR⁺EGFRt⁺ Line C occurred after 2 REM stimulations oftransduced CD8+T_(CM)-derived cells. Selection of CD19CAR⁺EGFRt⁺ Line Doccurred after 1 REM stimulation of transduced T_(EM)-derived cells.Selection of CD19CAR⁺EGFRt⁺IMPDH2dm⁺ Line E occurred after 1 REMstimulation of transduced T_(CM)-derived cells.

FIGS. 3a-b show that the EGFRt expressed on selected T cells is inert.In FIG. 3a , EGFRt expressed on T cells is not phosphorylated uponco-incubation with EGF. Negative control T cells, CD19CAR⁺EGFRt⁺ Line Acells, or A431 cells were incubated for 5 minutes with or without either100 ng/mL EGF or cetuximab (referred to in the figure as Erbtx) and thenlysed in the presence of phosphatase inhibitor. Lysates run on Westernblots were then probed using antibodies specific for either β-actin, thecytoplasmic domain of EGFR, or the phosphorylated tyrosine at position1068 of EGFR. FIG. 3b shows that EGF does not bind to the surface ofEGFRt expressing T cells. A431, Line A, and negative control T cellswere stained with PE-conjugated anti-EGFR, or either biotinylatedcetuximab or biotinylated EGF followed by PE-conjugated streptavidin(black histogram) versus PE-conjugated isotype control Ab orstreptavidin alone (open histogram) by flow cytometry. Percent positivestaining is indicated in each histogram.

FIGS. 4a-d illustrate that selected EGFRt⁺ CD19R⁺ T cells can beexpanded with maintenance of effector phenotype. FIG. 4a is a line graphshowing expansion of EGFRt-selected T cells, Lines A-E, over 12 or moredays after rapid expansion medium (REM) stimulation was initiated on theday of AutoMACS™ selection (day 0). (MACS is magnetic activated cellsorting.) Expansion of T cells in rapid expansion medium (REM) involvedthe incubation of 10⁶ T cells with 30 ng/mL anti-CD3ε (OKT3; OrthoBiotech, Raritan, N.J.), 5×10⁷ γ-irradiated PBMCs (3500 cGy), and 10⁷γ-irradiated LCLs (8000 cGy) in 50 mL CM; with addition of 50 U/mLrhIL-2 and 10 ng/ml rhIL-15 (CellGenix) every 48 hours, beginning onday 1. T cells were re-stimulated in this manner every 14 days. FIG. 4bshows histograms representing EGFRt-selected T cells (11 to 13 daysafter stimulation) that were phenotyped for surface EGFR (i.e., EGFRt,with biotinylated cetuximab), Fc (i.e., CAR), and T cell markers CD4 orCD8, (black histogram) vs. isotype control Ab (open histogram) by flowcytometry. Percent positive staining is indicated in each histogram.“N.D.” indicates no data. FIG. 4C are five lines graphs, one for each ofLines A-E, of EGFRt-selected T cells (within 11 to 15 days after REMstimulation) incubated for 4 hours with ⁵¹Cr-labled NS0, U251T,CD19t-expressing NS0, CMV pp65-expressing U251T, CD19-expressing Daudior SupB15, or OKT3-expressing LCL cells as targets at the indicated E:Tratios. Chromium release was measured to determine cytotoxic activity.FIG. 4d is a graph showing MPA resistance of the CD19CAR⁺EGFRt⁺IMPDH2dm⁺Line E. Control T cells that do not express IMPDH2dm and EGFRt-selectedIMPDH2dm-expressing Line E cells were cultured either with or without 1μM MPA and total cell numbers were monitored.

FIG. 5 shows EGFRt expression can be used as a tracking marker for invivo T cell engraftment. Day 36 bone marrow harvested from a controlmouse or from a mouse that had received 10⁷ CD19CAR⁺EGFRt⁺ Line C at day0 was stained using PerCP-conjugated anti-human CD45 and biotinylatedcetuximab (“Bio-Erb”) followed by PE-conjugated streptavidin. Quadrantswere created based on isotype control staining, and percent positivestaining in each quadrant is indicated in each histogram.

FIG. 6 is a graph showing EGFRt expression targets T cells for cetuximab(referred to in the figure as Erbitux®) mediated ADCC. ⁵¹Cr-labeled LineA cells were pre-incubated either with or without up to 20 μg/mL ofcetuximab or the CD20-specific mAb Rituxan as a negative control priorto addition of human PBMC as effectors.

FIG. 7 is the nucleotide (sense strand is SEQ ID NO: 1, antisense strandis SEQ ID NO: 2) and amino acid (SEQ ID NO: 3) sequences of GMCSFR alphachain signal sequence linked to EGFRt. The GMCSFR alpha chain signalsequence, which directs surface expression, is encoded by nucleotides1-66. EGFRt is encoded by nucleotides 67-1071.

FIG. 8 is the nucleotide (sense strand is SEQ ID NO: 4, antisense strandis SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences ofCD19R-CD28gg-Zeta(CO)-T2A-EGFRt. CD19R-CD28gg-Zeta(CO) is encoded bynucleotides 1-2040; T2A is encoded by nucleotides 2041-2112; GMCSFR isencoded by nucleotides 2113-2178; EGFRt is encoded by nucleotides2179-3186.

FIG. 9 is a graph showing CD19R-CD28gg-Zeta(CO)-T2A-EGFRt expression.Transduction of anti-CD3/anti-CD28 bead stimulated primary T cell blastswith the CD19R-CD28gg-Zeta(CO)-T2A-EGFRt_epHIV7 lentiviral vector(MOI=3) results in surface detection of both the CAR (using abiotinylated anti-Fc Ab and streptavidin-PE) and the truncated EGFRmolecule (using a biotinylated cetuximab Ab and streptavidin-PE) by flowcytometry on day 4. The white peak in each panel is non-transducedcontrol T cell blasts.

FIG. 10 is a schema showing a possible process flow for clinical trialsfor testing products of the present disclosure.

DETAILED DESCRIPTION

Certain embodiments of the invention are described in detail, usingspecific examples, sequences, and drawings. The enumerated embodimentsare not intended to limit the invention to those embodiments, as theinvention is intended to cover all alternatives, modifications, andequivalents, which may be included within the scope of the presentinvention as defined by the claims. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

Erbitux® is a registered trademark for the anti-EGFR monoclonal antibodycetuximab and is intended to independently include the trade nameproduct formulation, the generic drug, and the active pharmaceuticalingredient(s) of the trade name product.

The term “genetic modification” means any process that adds, deletes,alters, or disrupts an endogenous nucleotide sequence and includes, butis not limited to viral mediated gene transfer, liposome mediatedtransfer, transformation, transfection and transduction, e.g., viralmediated gene transfer such as the use of vectors based on DNA virusessuch as lentivirus, adenovirus, retroviruses, adeno-associated virus andherpes virus.

The term “antibody” includes monoclonal antibodies, polyclonalantibodies, dimers, multimers, multispecific antibodies and antibodyfragments that may be human, mouse, humanized, chimeric, or derived fromanother species. A “monoclonal antibody” is an antibody obtained from apopulation of substantially homogeneous antibodies that is beingdirected against a specific antigenic site.

“Variant” refers to polypeptides having amino acid sequences that differto some extent from a native sequence polypeptide. Ordinarily, aminoacid sequence variants will possess at least about 80% sequenceidentity, more preferably, at least about 90% homologous by sequence.The amino acid sequence variants may possess substitutions, deletions,and/or insertions at certain positions within the reference amino acidsequence.

“Percentage identity” or “percent identity” is defined as the percentageof residues in the amino acid sequence variant that are identical afterbest aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Methods and computerprograms for the alignment are well known in the art. Such programsinclude GAP, BESTFIT, FASTA, BLAST or Align 2.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors, such as natural killer cells, neutrophils, andmacrophages, recognize bound antibody on a target cell and cause lysisof the target cell. ADCC activity may be assessed using methods, such asthose described in U.S. Pat. No. 5,821,337.

“Effector cells” are leukocytes which express one or more constantregion receptors and perform effector functions.

To “treat” a disease or a disorder, such as cancer, means to take eithertherapeutic measures or preventative measures to lessen or abate thedisease or disorder. Such treatment includes prevention, alleviation ofsymptoms, diminishment or stabilization of scope, and/or remission.

The term “therapeutically effective amount” refers to an amount of acompound or molecule effective to treat a disease or disorder.

“Cancer” refers to cells undergoing uncontrolled cellular growth.Examples of cancer include colorectal cancer and head and neck cancer. A“chemotherapeutic agent” is a chemical compound useful in the treatmentof cancer.

A “cytokine” is a protein released by one cell to act on another cell asan intercellular mediator.

“Non-immunogenic” refers to a material that does not initiate, provokeor enhance an immune response where the immune response includes theadaptive and/or innate immune responses.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons). Some genes may bedeveloped which lack, in whole or in part, introns. Some leadersequences may enhance translation of the nucleic acid into polypeptides.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

As used herein, a “vector” may be any agent capable of delivering ormaintaining nucleic acid in a host cell, and includes viral vectors(e.g. retroviral vectors, lentiviral vectors, adenoviral vectors, oradeno-associated viral vectors), plasmids, naked nucleic acids, nucleicacids complexed with polypeptide or other molecules and nucleic acidsimmobilized onto solid phase particles. The appropriate DNA sequence maybe inserted into the vector by a variety of procedures. In general, theDNA sequence is inserted into an appropriate restriction endonucleasesite(s) by procedures known in the art. Such procedures and others aredeemed to be within the scope of those skilled in the art. Transcriptionof the DNA encoding the polypeptides of the present invention by highereukaryotes is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase its transcription. Examplesincluding the SV40 enhancer on the late side of the replication originbp 100 to 270, a cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

“Receptor” means a polypeptide that is capable of specific binding to amolecule. Whereas many receptors may typically operate on the surface ofa cell, some receptors may bind ligands when located inside the cell(and prior to transport to the surface) or may reside predominantlyintra-cellularly and bind ligand therein.

“Antibody or functional fragment thereof” means an immunoglobulinmolecule that specifically binds to, or is immunologically reactive witha particular antigen or epitope, and includes both polyclonal andmonoclonal antibodies. The term antibody includes genetically engineeredor otherwise modified forms of immunoglobulins, such as intrabodies,peptibodies, chimeric antibodies, fully human antibodies, humanizedantibodies, and heteroconjugate antibodies (e.g., bispecific antibodies,diabodies, triabodies, and tetrabodies). The term functional antibodyfragment includes antigen binding fragments of antibodies, includinge.g., Fab′, F(ab′).sub.2, Fab, Fv, rIgG, and scFv fragments. The termscFv refers to a single chain Fv antibody in which the variable domainsof the heavy chain and of the light chain of a traditional two chainantibody have been joined to form one chain.

In one embodiment, a gene encoding a modified endogenous cell-surfacemolecule that may be used as a non-immunogenic selection epitopecompatible with immunomagnetic selection is provided. Such anon-immunogenic selection epitope may facilitate immunotherapy in cancerpatients without undesirable immunologic rejection of cell products. Theendogenous cell surface molecule may be modified or truncated to retainan extracellular epitope recognized by a known antibody or functionalfragment thereof, and to remove any signaling or trafficking domainsand/or any extracellular domains unrecognized by said known antibody. Amodified endogenous cell surface molecule which lacks a signaling ortrafficking domain and/or any extracellular domains unrecognized by saidknown antibody is rendered inert.

The modified endogenous cell-surface molecule may be, but is not limitedto, any non-immunogenic cell-surface related receptor, glycoprotein,cell adhesion molecule, antigen, integrin or cluster of differentiation(CD) that is modified as described herein. Modification of suchcell-surface molecules is accomplished by keeping an epitope that isrecognized by a known antibody or functional fragment thereof; andremoving any signaling or trafficking domains and/or any extracellulardomains unrecognized by a known antibody. Removal of the signaling ortrafficking domains and/or any extracellular domains unrecognized by aknown antibody renders the endogenous cell-surface moleculenon-immunogenic and/or inert.

Examples of endogenous cell-surface molecules that may be modified ortruncated according to the embodiments described herein include, but arenot limited to EpCAM, VEGFR, integrins (e.g., integrins ανβ3, α4,αIIbβ3, α4β7, α5β1, ανβ3, αν), TNF receptor superfamily (e.g., TRAIL-R1,TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB,ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3,or clusters of differentiation (e.g., CD2, CD3, CD4, CD5, CD11,CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/IgE Receptor,CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74,CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5,CD319/SLAMF7).

Corresponding commercial antibodies that may be used to recognize amodified or truncated endogenous cell-surface molecule include, but arenot limited to, 3F8, abagovomab, abciximab, adecatumumab, afutuzumab,alemtuzumab, altumomab pentetate, anatumomab mafenatox, apolizumab,arcitumomab, aselizumab, atlizumab (=tocilizumab), basiliximab,bectumomab, benralizumab, besilesomab, bivatuzumab mertansine,blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromabpendetide, catumaxomab, CC49, cedelizumab, celmoleukin, citatuzumabbogatox, clenoliximab, clivatuzumab tetraxetan, CNTO-95, conatumumab,dacetuzumab, daclizumab, daratumumab, detumomab, ecromeximab,edrecolomab, efalizumab, elotuzumab, enlimomab pegol, epitumomabcituxetan, epratuzumab, erlizumab, etaracizumab, fanolesomab,faralimomab, farletuzumab, galiximab, gavilimomab, gemtuzumabozogamicin, glembatumumab vedotin, gomiliximab, ibalizumab, ibritumomabtiuxetan, igovomab, intetumumab, iratumumab, inolimomab, inotuzumabozogamicin, ipilimumab, keliximab, labetuzumab, lintuzumab, lexatumumab,lucatumumab, lumiliximab, mapatumumab, maslimomab, milatuzumab,minretumomab, mitumomab, muromonab-CD3, naptumomab estafenatox,natalizumab, ocrelizumab, odulimomab, ofatumumab, olaratumab,oportuzumab monatox, oregovomab, otelixizumab, pemtumomab, priliximab,PRO 140, rituximab, rovelizumab, ruplizumab, satumomab pendetide,siplizumab, sontuzumab, tadocizumab, taplitumomab paptox, teneliximab,teplizumab, TGN1412, ticilimumab (=tremelimumab), tigatuzumab,tocilizumab (=atlizumab), toralizumab, tositumomab, tremelimumab,tucotuzumab, vedolizumab, veltuzumab, visilizumab, vitaxin, volociximab,votumumab, zanolimumab, ziralimumab, zolimomab aritox.

In some embodiments, the modified endogenous cell-surface molecule isencoded by a modified or truncated tyrosine kinase receptor gene.Examples of tyrosine kinase receptors that may be modified or truncatedaccording to the embodiments described herein include, but are notlimited to, members of the endothelial growth factor receptor family(EGRF/ErbB1/HER1; ErbB2/HER2/neu; ErbB3/HER3; ErbB4/HER4), hepatocytegrowth factor receptor (HGFR/c-MET) and insulin-like growth factorreceptor-1 (IGF-1R). According to some embodiments, modified tyrosinekinase receptors retain an extracellular epitope recognized by a knownantibody or functional fragment thereof, and lack at least a tyrosinekinase domain. A modified tyrosine kinase receptor which lacks at leasta tyrosine kinase domain renders the receptor inert.

Commercial antibodies that may be used to recognize a modified tyrosinekinase receptor include, but are not limited to AMG-102, AMG-479,BIIB022OA-5D5, CP-751,871, IMC-A12, R1507, cetuximab, cixutumumab,ertumaxomab, figitumumab, matuzumab, necitumumab, panitumumab,pertuzumab, nimotuzumab, robatumumab, trastuzumab, zalutumumab.

In one embodiment, the modified endogenous cell surface molecule is atruncated EGFR (tEGFR) that lacks the membrane distal EGF-binding domainand the cytoplasmic signaling tail, but retains the extracellularmembrane proximal epitope recognized by a known antibody or functionalfragment thereof (e.g., cetuximab, matuzumab, necitumumab orpanitumumab). In another embodiment, the tEGFR is missing Domain I,Domain II, the Juxtamembrane Domain and the Tyrosine Kinase Domain ascompared to an unmodified EGFR (FIG. 1).

A gene encoding a modified endogenous cell surface molecule may be usedas a cell selection or enrichment marker for a genetically modifiedpopulation of immune cells (e.g., T cells). The gene encoding a modifiedendogenous cell surface molecule may be coupled to a gene encoding atumor targeting chimeric antigen receptor (CAR). These genes may beinserted into a vector to transduce the population of T cells to begenetically modified. After transduction, the cells that aresuccessfully transduced and express the CAR and modified endogenouscell-surface molecule are enriched by any suitable purification method,such as immunomagnetic purification with anti-biotin microbeads orfluorochrome-conjugated anti-biotin for fluorescence activated cellsorting, using a commercial antibody that recognizes the modifiedendogenous cell-surface molecule expressed by the transduced cell.

In another embodiment, a gene encoding a truncated human epidermalgrowth factor receptor (EGFRt) that lacks the membrane distalEGF-binding domain and the cytoplasmic signaling tail, but retains theextracellular membrane proximal epitope recognized by the FDA-approvedanti-EGFR monoclonal antibody (mAb) cetuximab or another anti-EGFRantibody, is constructed and described herein. The EGFRt may be coupledwith chimeric antigen receptors specific for a tumor associated antigen.The tumor associated antigen may be CD19, CD20, or CD22, or any othertumor associated antigen, but is preferably CD19 (CD19CAR). The tumorassociated antigen is followed by a C-terminal 2A cleavable linker andthe coding sequence for EGFRt. The biotinylated-cetuximab may be used inconjunction with commercially available anti-biotin microbeads for thepurpose of immunomagnetic purification of the tumor associatedantigen/CAR-expressing transductants. In the instance where the tumorassociated antigen is CD19 the product is CD19CAR-expressingtransductants. Alternatively, the biotinylated-cetuximab may be used inconjunction with Fluorochrome-conjugated anti-biotin for fluorescenceactivated cell sorting.

In another embodiment, a modified endogenous cell-surface molecule maybe used as a marker for in vivo T cell engraftment. For example, whenthe modified endogenous cell-surface molecule is EGFRt, the EGFRt may beused to track the uptake of the T cells to which it is attached in vivowithout affecting cellular function of the T cells or the cells to whichthe T cells are targeted, such as bone marrow cells in a transplantsituation. The use of cetuximab conjugated to probes or reporter genessuch as sr39TK may be used to improve the tracking potential ofEGFRt-expressing cells to patients via PET imaging techniques.

In a separate embodiment, a modified endogenous cell-surface moleculemay be used to induce cell suicide. For example, EGFRt may be used as asuicide gene via cetuximab mediated complement and/or antibody dependentcell mediated cytotoxicity (ADCC) pathways. The fact that cetuximab is atherapeutic FDA-approved antibody further facilitates the suicide genepotential of EGFRt in the clinical setting.

In other embodiments, the truncated epidermal growth factor receptor(EGFRt) selection epitope or other modified cell-surface molecule isattached to other sequences. One exemplar sequence is the GMCSFR alphachain signal sequence, which directs surface expression, attached toEGFRt. GMCSFR is encoded by nucleotides 1-66 and EGFRt is encoded bynucleotides 67-1071 of SEQ ID NO: 1. See FIG. 7. Also in FIG. 7 is theantisense strand (SEQ ID NO: 2) and amino acid (SEQ ID NO: 3) sequencesof GMCSFR alpha chain signal sequence linked to EGFRt. Another suchsequence is a codon-optimized cDNA sequence encoding an anti-CD19costimulatory chimeric antigen receptor (CD19R-CD28gg-Zeta(CO)), and acleavable T2A linker. Cytotoxic T lymphocytes (CTLs) modified to expressa CD19-specific chimeric antigen receptor (CAR) that signals via acytoplasmic costimulatory (CD28) domain fused to the cytoplasmic CD3-ζdomain exhibits superior anti-tumor potency that can be attributed toCD28-mediated survival and enhanced cytokine production. This constructmay be further modified to incorporate a C-terminal 2A cleavable linkerfollowed by the coding sequence for a truncated human EGFR (EGFRt) forthe purpose of immunomagnetic purification of CAR-expressingtransductants using cetuximab-biotin/anti-biotin microbeads. See theCD19R-CD28gg-Zeta(CO)-T2A-EGFRt sequence attached as FIG. 8, SEQ ID NOS:4 (nucleotide sense strand), 5 (nucleotide antisense strand), and 6(protein). Lentivector transduction of primary human T cells with thiscodon-optimized cDNA directs the coordinated expression of the CAR andEGFRt (FIG. 9).

To eliminate variability between transgene expression products otherwiseintrinsic to transduction procedures without subsequent selection, anon-immunogenic selection epitope, EGFRt, compatible with immunomagneticselection using the CliniMACS device (Miltenyi Biotec, BergischGladbach, Germany) was developed. For example, EGFRt is a truncatedhuman epidermal growth factor receptor that lacks the membrane distalEGF-binding domain and the ectoplasmic signaling tail, but retains theextracellular membrane proximal epitope recognized by the commercialanti-EGFR mAb cetuximab. See FIG. 1. Biotinylated-cetuximab is appliedto immunomagnetic selection in combination with anti-biotin microbeads(Miltenyi). Human OKT3 blasts that had been lentivirally transduced withCD19R-CD28gg-Zeta(CO)-T2A-EGFRt were subjected to immunomagneticselection using the Miltenyi AutoMACS device, and the frequency ofEGFRt+CAR+ T cells was enriched from 22% (pre-selection) to 99%(post-selection) without observable toxicity to the cell preparation. Itis also possible that, instead of or in addition to immunomagneticsorting, the EGFRt can be purified using fluorescence-based cell sortingtechniques.

Due to the absence of the EGF-binding domains and intracellularsignaling domains, EGFRt is inactive when expressed by T cells.Importantly, the EGFRt-selected T cells maintain their desired effectorphenotype—including anti-tumor cyotoxic activity mediated by thechimeric antigen receptor that is coordinately expressed with theEGFRt—and remain amenable to established expansion protocols.

Overall, this EGFRt has various advantages for immunotherapeutic cellproducts compared to other selection markers that have been previouslyreported. Specifically, unlike truncated CD4 and CD19, it is notendogenously expressed by subpopulations of lymphocytes. Furthermore, incontrast to truncated CD34 and low affinity nerve growth factorreceptor, it does not have any activity that might negatively affect theimmune cell product (i.e., in terms of signaling or trafficking).Lastly, it alone can be bound/recognized by a known, preferablycommercially available, pharmaceutical grade antibody reagent, i.e.,cetuximab. Together, these attributes make EGFRt a superior selectionmarker for any transfection/transduction system that can be applied tothe generation of cell products for adoptive immunotherapy. Thus, EGFRtis well suited to be used as a selection marker for lentivirallytransduced T cells of immunotherapeutic relevance.

Also provided are methods for identifying new therapeutic cell productshaving the following criteria: a modified endogenous cell-surfacemolecule, ligand or receptor that is not, as modified, endogenouslyexpressed in the subject in which it is intended to be therapeuticallyutilized, does not have any immunoactivity or other functional activitythat would hinder the functioning of the product or the subject intowhich the product is administered, and that it can be recognized by aknown antibody.

Having described the invention with reference to the embodiments andillustrative examples, those in the art may appreciate modifications tothe invention as described and illustrated that do not depart from thespirit and scope of the invention as disclosed in the specification. Theexamples are set forth to aid in understanding the invention but are notintended to, and should not be construed to limit its scope in any way.The examples do not include detailed descriptions of conventionalmethods. Such methods are well known to those of ordinary skill in theart and are described in numerous publications.

Example 1: Generation of EGFRt and Immunomagnetic Selection of EGFRtExpressing T Cells

Materials & Methods

Antibodies and Flow Cytometry

FITC-, PE- and PerCP-conjugated isotype controls, PerCP-conjugatedanti-CD8, FITC conjugated anti-CD4, PE-conjugated anti-IFNγ,PerCP-conjugated anti-CD45 and PE-conjugated streptavidin were obtainedfrom BD Biosciences (San Jose, Calif.). Biotinylated anti-Fc waspurchased from Jackson ImmunoResearch Laboratories, Inc. (Westgrove,Pa.). PE-conjugated anti-Biotin was purchased from Miltenyi Biotec(Auburn, Calif.). Biotinylated EGF was purchased from Molecular Probes®Invitrogen (Carlsbad, Calif.). PE-conjugated anti-EGFR was purchasedfrom Abcam Inc. (Cambridge, Mass.). All antibodies and biotin-EGF wereused according to the manufacturer's instructions. Flow cytometric dataacquisition was performed on a FACScalibur (BD Biosciences), and thepercentage of cells in a region of analysis was calculated using FCSExpress V3 (De Novo Software, Los Angeles, Calif.).

For generation of the biotinylated-cetuximab, 200 mg of cetuximab(Erbitux®) was buffer exchanged (19 hours) to PBS (D-PBS, pH 7.5±0.1)using a MidGee Hoop Cartridge (UFP-30-E-H42LA) with 527 mL. The materialat 2 mg/mL was then modified at a 20:1 ratio using Sulfo-NHS-LC-Biotinin a reaction that was carried out for 1 hour at room temperature andthen diafiltered to remove the excess biotin. The 200 mg of biotinylatedcetuximab was then buffer exchanged (18 hours) to PBS (D-PBS, pH7.5±0.1) using MidGee Hoop Cartridge (UFP-30-E-H42LA) with 533 mL.Glycerol was added to a final concentration of 20% and then the materialwas frozen in vials.

Cell Lines

Unless otherwise indicated, all cell lines were maintained in RPMI 1640(Irvine Scientific, Santa Ana, Calif.) supplemented with 2 mML-glutamine (Irvine Scientific), 25 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, IrvineScientific), 100 U/mL penicillin, 0.1 mg/mL streptomycin (IrvineScientific), and 10% heat-inactivated fetal calf serum (FCS, Hyclone,Logan, Utah), hereafter referred to as culture media (CM).

To generate T cells, human peripheral blood mononuclear cells (PBMC)were isolated by density gradient centrifugation over Ficoll-Paque(Pharmacia Biotech, Piscataway, N.J.) from heparinized peripheral bloodobtained from consented healthy donors participating on a City of HopeNational Medical Center Internal Review Board-approved protocol. Forgeneration of Line A, washed PBMC were stimulated with 25 U/mL IL-2 anda 1:1 (cell:bead) ratio of Dynabeads® Human T expander CD3/CD28(Invitrogen, Carlsbad, Calif.). For generation of the other lines,washed PBMC were first autoMACS™ depleted using anti-CD45RA beads(Miltenyi Biotec) per the manufacturer's protocol, and in some casesalso depleted with PE-conjugated anti-CD4 (BD Biosciences) with anti-PEbeads (Miltenyi Biotec). The resulting cells then underwent autoMACS™positive selection using biotinylated DREG56 (anti-CD62L) andanti-biotin beads (Miltenyi Biotec) to produce purified CD62L⁺CD45RO⁺T_(CM). CD8⁺ cells were further selected in some cases using AutoMACS™(Miltenyi Biotec) per the manufacturer's protocol. CMV-specific cellswere generated by stimulating T cells with 5 U/ml rhIL-2 (Chiron,Emeryville, Calif.) and autologous irradiated viral antigen presentingcells at a 4:1 (responder:stimulator) ratio once a week for three weeks,using 10% human serum instead of FCS to avoid non-specific stimulation.The viral antigen presenting cells were derived from PBMC that had beengenetically modified to express CMVpp65 antigen.

PBMC were resuspended in nucleofection solution using the Human T cellNucleofector kit (Amaxa Inc., Gaithersberg, Md.), and 5×10⁷ cells werealiquoted into 0.2-cm cuvettes containing 10 μg HygroR-pp65_pEK (orpmaxGFP from Amaxa Inc., as a transfection control) in a final volume of100 μL/cuvette, and electroporated using the Amaxa Nucleofector I (AmaxaInc.), program U-14, after which cells were allowed to recover for 6hours at 37° C. prior to γ-irradiation (1200 cGy).

The CD19CAR-T2A-EGFRt_epHIV7 (pJ02104) andCD19CAR-T2A-EGFRt-T2A-IMPDH2dm_epHIV7 (pJ02111) lentiviral constructscontain a) the chimeric antigen receptor (CAR) sequences consisting ofthe V_(H) and V_(L) gene segments of the CD19-specific FmC63 mAb, anIgG1 hinge-C_(H2)-C_(H3), the transmembrane and cytoplasmic signalingdomains of the costimulatory molecule CD28, and the cytoplasmic domainof the CD3ζ chain[10]; b) the self-cleaving T2A sequence[11]; c) thetruncated EGFR sequence (See FIG. 1); and d) the IMPDH2 double mutantthat confers MPA-resistance, as indicated. Lentiviral transduction wascarried out on T cells that were stimulated with either 30 ng/mLanti-CD3ε (OKT3; Ortho Biotech, Raritan, N.J.) (i.e., for Line A) orhuman CD3/CD28Dynal beads at a 1:10 ratio (i.e., for Lines B, C, D andE) and 25 U IL2/ml. Cells were cultured for up to 2 hours at 37° C. onRetroNectin® (50 ug/ml) coated plates prior to addition of thelentivirus at an MOI of 3 and 5 μg/ml polyybrene. After 4 hours, warmmedium was added to triple to volume, and the cells were then washed andplated in fresh media after 48 hours. AutoMACS™ sorting ofEGFRt-expressing cells was carried out with biotinylated cetuximab andanti-biotin microbeads (Miltenyi Biotec) as per the manufacturer'sinstructions. Expansion of T cells in rapid expansion medium (REM)involved the incubation of 10⁶ T cells with 30 ng/mL anti-CD3ε (OKT3;Ortho Biotech, Raritan, N.J.), 5×10⁷ γ-irradiated PBMCs (3500 cGy), and10⁷ γ-irradiated LCLs (8000 cGy) in 50 mL CM; with addition of 50 U/mLrhIL-2 and 10 ng/ml rhIL-15 (CellGenix) every 48 hours, beginning onday 1. T cells were re-stimulated in this manner every 14 days.

EBV-transformed lymphoblastoid cell lines (LCLs) were made from PBMC aspreviously described [13]. LCL-OKT3 cells were generated by resuspendingLCL in nucleofection solution using the Amaxa Nucleofector kit T, addingOKT3-2A-Hygromycin_pEK (pJ01609) plasmid at 5 μg/10⁷ cells, andelectroporating cells using the Amaxa Nucleofector I, program T-20. Theresulting LCL—OKT3-2A-Hygro_pEK (cJ03987) were grown in CM containing0.4 mg/ml hygromycin. The mouse myeloma line NS0 (gift from AndrewRaubitschek, City of Hope National Medical Center, Duarte, Calif.) wasresuspended in nucleofection solution using the Nucleofector kit T(Amaxa Inc., Gaithersberg, Md.), CD19t-DHFRdm-2A-IL12_pEK (pJ01607) orGFP-IMPDH2dm-2A-IL15_pcDNA3.1(+) (pJ01043) plasmid was added at 5μg/5×10⁶ cells, and cells were electroporated using the AmaxaNucleofector I, program T-27. The resulting NS0—CD19t-DHFRdm-2A-IL12_pEK(cJ03935) and NS0—GFP:IMPDH2-IL15(IL2ss)_pcDNA3.1(+) (cJ02096) weregrown in DMEM (Irvine Scientific, Santa Ana, Calif.) supplemented with10% heat-inactivated FCS, 25 mM HEPES, and 2 mM L-glutamine in thepresence of either 0.05 uM methotrexate (MTX) or 6 μM mycophenolic acid(MPA). The tumorigenic strain of U251, termed U251T, was a kind gift ofDr. Waldemar Debinski (Wake Forest, N.C.). U251T-pp65 were generated bylentiviral transduction of U251T with pp65-2A-eGFP-ffluc_epHIV7(pJ01928) at an MOI of 1. The resulting U251T—pp65-2A-eGFP-ffluc_epHIV7were then FACS sorted for the GFP⁺ population (cJ05058). The Daudilymphoma line was purchased from ATCC and grown in media consisting ofRPMI 1640 (Irvine Scientific), 2 mM L-Glutamine (Irvine Scientific), 10%heat-inactivated FCS (Hyclone). SupB15 acute lymphoblastic leukemiacells and A431 epidermoid carcinoma cells were purchased from ATCC.

Protein Analysis

Cells (up to 10⁷) were lysed with 804 of 1% Triton-X lysis buffercontaining phosphatase inhibitor cocktail II (Sigma-Aldrich Corp., St.Louis, Mo.) (1:20 of inhibitor to buffer by volume). 50 μg of proteinwas loaded in each lane, and Western blots were probed with antibodiesfrom the Phospho-EGF receptor antibody sampler kit (Cell SignalingTechnology, Inc., Danvers, Mass.) followed by IRDye™ 680 CW or 800CWconjugated goat anti-rabbit antibodies (LI-COR, Lincoln, Nebr.), as wellas the IRDye™ 800 conjugated anti-beta-Actin antibody (LI-COR) as perthe manufacturers' instructions. Blots were imaged on the OdysseyInfrared Imaging System (LI-COR).

Chromium-Release Assays

The cytolytic activity of T cells was determined by 4-hourchromium-release assay (CRA), where effector cells were seeded intotriplicate wells of V-bottom 96-well micro-plates containing 5×10³⁵¹Cr-labeled targeT cells (Na₂ ⁵¹CrO₄; (5 mCi/mL); Amersham Pharmacia,Piscataway, N.J.) at various E:T ratios in 200 uL of CM and incubatedfor 4 hours at 5% CO₂, 37° C. Plates were centrifuged, and 100 μl ofsupernatant was removed from each well to assess chromium release usinga γ-counter (Packard Cobra II, Downer's Grove, Ill.). The percentspecific lysis was calculated as follows: 100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release).Maximum release was determined by measuring the ⁵¹Cr content of wellscontaining labeled targets lysed with 2% SDS.

Antibody dependent cell mediated cytotoxicity was determined by chromiumrelease as above using 5×10³ ⁵¹Cr-labeled targeT cells that had beenpre-incubated for 90 min with up to 10 μg/mL of either cetuximab orrituximab (a CD20-specific mAb), washed and then co-incubated with 5×10⁵freshly isolated PBMC.

T Cell Engraftment and Cetuximab Mediated Suicide In Vivo

For T cell engraftment, six- to ten-week old NOD/Scid IL-2RγC^(null)mice are injected i.v. on day 0 with 10⁷ T cells (Line C). 2×10⁷irradiated (8000 rads) NS0—GFP:IMPDH2-IL15(IL2ss)_pcDNA3.1(+) (cJ02096)cells are administered i.p. 3 times a week starting on day 0 to providea systemic supply of human IL-15 in vivo. Bone marrow was harvested fromeuthanized animals and analyzed by flow cytometry. Antibody dependentcell mediated cytotoxicity assays are performed to determine theactivity of cetuximab against EGFRt⁺ T cells.

Results

Immunomagnetic Selection of EGFRt Expressing T Cells

A truncated human EGFR (EGFRt), which contains only the transmembranedomain and extracellular domains III and IV of the full length EGFR, wasgenerated as a non-immunogenic selection epitope compatible withimmunomagnetic selection. As shown in the FIG. 1 molecular model, theEGFRt retains the ability to be bound by cetuximab, but not have anysignaling capacity due to the absence of the intracellular domains.Furthermore, it lacks the N-terminal domain required for EGF-binding.

To immunomagnetically select for EGFRt-expressing cells,biotinylated-cetuximab was generated (FIG. 2a, b ) to be used inconjunction with commercially available anti-biotin microbeads and anAutoMACS™ separator (Miltenyi Biotec) (FIG. 2c ). Lentiviraltransduction of various T cell lines with EGFRt-containing constructs,where the EGFRt gene was separated from other genes of interest oneither one or both ends with the self-cleaving T2A sequence,consistently resulted in surface detection of the EGFRt molecule on lessthan 40% of the cells (FIG. 2d ). Surface detection may also beaccomplished with a EGFRt-sr39TK fusion. Immunomagnetic selectionallowed for recovery of EGFRt⁺ T cell populations with greater than 90%purity. T cell populations that underwent this transduction andselection procedure included anti-CD3/anti-CD28 bead stimulated T cellblasts (for Line A), central memory (CD45RO⁺CD62L⁺ T_(CM)) derived Tcells (for Lines B, C and E), which in some cases were also pre-selectedfor CMV specificity (via the endogenous TCR; for Line B) or CD8expression (for Line C), as well as effector memory (CD62L⁻ CD45RO⁺T_(EM)) derived T cells (for line D). These data show that EGFRt cansuccessfully be used as a selection marker for various sources of T celltransductants, even when the original transduction efficiency was as lowa 2%.

Inactivity of EGFRt on Selected T Cells

To confirm that the EGFRt is inactive, Western immunoblot analyses forEGFR phosphorylation were carried out on the EGFRt-selected T cellsafter culture with either EGF or cetuximab. As expected, cetuximab didnot induce EGFR phosphorylation above background even in the EGFR⁺ cellline A431 (FIG. 3a ). Furthermore, in contrast to that seen with theA431 cells, no phosphorylation was seen in lysates of Line A afterco-incubation with EGF. Indeed, using biotinylated EGF, flow cytometricanalysis confirmed that EGF cannot bind the EGFRt-selected T cells (FIG.3b ), as expected due to the truncation in its N-terminus. These EGFRt⁺T cells were also not recognized by another anti-EGFR antibody distinctfrom cetuximab.

Maintenance of Effector Phenotype in Expanded EGFRt⁺ CD19CAR⁺ T Cells

Directly after AutoMACS™ separation, the selected T cells were expanded30-fold or greater within 12 days after REM stimulation with OKT3,irradiated PBMC feeders and LCL, IL-2 and IL-15 (FIG. 4a ). Flowcytometric analysis of the resulting expanded EGFRt⁺ T cells furtherconfirmed that that they express the CD19CAR and T cell markers such asCD8, TCR, CD3, perforin, granzyme, etc. (FIG. 4b ). Furthermore,CD19CAR-directed cytotoxic activity of these EGFRt-selected lines isevident in chromium release assays using CD19-expressing tumor targets(FIG. 4c ). A direct comparison of the CD19-specific reactivity of LineE versus its non-selected or ‘parental’ counterpart shows that there isenhanced CD19CAR-mediated cytotoxicity upon EGFRt-selection. Inaddition, the CMV-specific T_(CM)-derived CD19CAR⁺EGFRt⁺ Line B cellsalso show cytotoxic activity through their endogenous T cell receptoragainst targets expressing CMV-pp65 antigen.

For the CD19CAR⁺EGFRt⁺IMPDH2dm⁺ Line E, the ability of the inosinemonophosphate dehydrogenase 2 double mutant (IMPDH2dm) to conferresistance to the IMPDH2-inhibitor mycophenolic acid (MPA; a commonimmunosuppressant used to prevent rejection in organ transplantation)was also tested. Upon culture in 1 uM MPA, the survival and/orproliferation of Line E cells is not inhibited (FIG. 4d ). This is incontrast to the inhibition seen with a control T cell line that lacksexpression of the IMPDH2dn gene. These data provide further evidencethat EGFRt-mediated selection results in the corresponding selection ofthe other genes present in the lentiviral construct used to transduce Tcells.

Tracking of EGFRt⁺ T Cells In Vivo

To test the potential for detecting in vivo engrafted T cells, bonemarrow cells collected from mice that had been engrafted withCD19CAR⁺EGFRt⁺ Line C was analyzed by flow cytometry using biotinylatedcetuximab (FIG. 5). Control mice that did not receive T cells revealedthat there was some cross-reaction of the cetuximab against murine EGFR.Thus, it was determined that successful detection of engrafted Line Ccells required double staining for both human CD45 and EGFRt. Cells mayalso analyzed using immunohistochemistry to determine potential forscreening biopsy material.

Cetuximab Mediated Cytotoxicity of EGFRt⁺ T Cells

Because cetuximab is known to lyse EGFR-expressing cells via antibodydependent cell mediated cytotoxicity (ADCC), assays were performed todetermine the ADCC activity of cetuximab against EGFRt⁺ T cells (FIG.6). Using ⁵¹Cr-labeled Line A cells as targeted and freshly isolatedhuman PBMC as effectors, cetuximab was found to significantly mediatechromium-release above that seen when using the CD20-specific humanizedmAb Rituxan.

Example of Therapeutic Use of EGFRt⁺ T Cells

Adult subjects with high-risk intermediate grade B-cell lymphomas whoare candidates for an autologous myeloablative stem cell transplantprocedure may receive post-transplant immunotherapy with adoptivelytransferred autologous Tcm-derived CD19R⁺ CD8⁺ EGFRt⁺ T cell grafts. Aleukapheresis product collected from each patient undergoes selection ofTcm, transduction with clinical grade CD19CAR-T2A-EGFRt_epHIV7, and thenselection and expansion of the EGFRt⁺ cells in a closed system. Afterthe resulting cell products have undergone quality control testing(including sterility and tumor specific cytotoxicity tests), they arecryopreserved. Meanwhile, following leukapheresis, study participantscommence with standard salvage chemotherapy, with mobilization for autoHSC collection with cytoreductive chemotherapy and G-CSF. Since theEGFRt-selected, CD19-specific T cells will also target normal CD20⁺(CD19⁺) B cells, the B cell numbers can first be lowered usingRituximab™ to reduce the recipient's inflammatory response uponreceiving the genetically modified CTL and also increase availability ofinfused T cells to immediately target lymphoma cells. Furthermore,Rituximab™ may blunt a humoral immune response against the geneticallymodified T cells. If Rituximab™ is not given as part of theSalvage/Priming chemotherapy regimen, research participants may receivea single intravenous infusion of Rituximab™ (chimeric anti-CD20antibody) at 375 mg/m² within 4-weeks of the planned auto-HSCTprocedure. Rituximab™ infusion would be carried out per standardpractice including premedication with diphenhydramine and acetaminophenand hydrocortisone. On Day +2 or Day +3 after HSCT, the autologouscryopreserved CD19R⁺ CD8⁺ EGFRt⁺ T cell product will be transported,thawed and infused at the patient's bedside. Research participants canbe pre-medicated at least 30 minutes prior to T cell infusion with 15mg/kg of acetaminophen P.O. (max. 650 mg.) and diphenhydramine 0.5-1mg/kg I.V. (max dose 50 mg). Clinical and laboratory correlativefollow-up studies can then be performed at the physician's discretion,and may include quantitative RT-PCR studies for the presence ofCD19-expressing lymphoma cells and/or the adoptively transfered T cells;FDG-PET and/or CT scans; bone marrow examination for disease specificpathologic evaluation; lymph node biopsy; and/or long-term follow up perthe guidelines set forth by the FDA's Biologic Response ModifiersAdvisory Committee that apply to gene transfer studies. FIG. 10 providesa possible schematic for clinical testing of the present products andmethods.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

All patents, patent applications, and references cited throughout thespecification are expressly incorporated by reference.

REFERENCES

-   1. Berger, C, Flowers, M E, Warren, E H, and Riddell, S R (2006).    Analysis of transgene-specific immune responses that limit the in    vivo persistence of adoptively transferred HSV-TK-modified donor T    cells after allogeneic hematopoietic cell transplantation. Blood    107: 2294-302.-   2. Tey, S K, Dotti, G, Rooney, C M, Heslop, H E, and Brenner, M K    (2007). Inducible caspase 9 suicide gene to improve the safety of    allodepleted T cells after haploidentical stem cell transplantation.    Biol Blood Marrow Transplant 13: 913-24.-   3. Fehse, B, Richters, A, Putimtseva-Scharf, K, Klump, H, Li, Z,    Ostertag, W, et al. (2000). CD34 splice variant: an attractive    marker for selection of gene-modified cells. Mol Ther 1: 448-56.-   4. Gaines, P, and Wojchowski, D M (1999). pIRES-CD4t, a dicistronic    expression vector for MACS- or FACS-based selection of transfected    cells. Biotechniques 26: 683-8.-   5. Fehse, B, Uhde, A, Fehse, N, Eckert, H G, Clausen, J, Ruger, R,    et al. (1997). Selective immunoaffinity-based enrichment of CD34+    cells transduced with retroviral vectors containing an    intracytoplasmatically truncated version of the human low-affinity    nerve growth factor receptor (deltaLNGFR) gene. Hum Gene Ther 8:    1815-24.-   6. Lemoine, F M, Mesel-Lemoine, M, Cherai, M, Gallot, G, Vie, H,    Leclercq, V, et al. (2004). Efficient transduction and selection of    human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector    produced by a human packaging cell line. J Gene Med 6: 374-86.-   7. Li, S, Schmitz, K R, Jeffrey, P D, Wiltzius, J J, Kussie, P, and    Ferguson, K M (2005). Structural basis for inhibition of the    epidermal growth factor receptor by cetuximab. Cancer Cell 7:    301-11.-   8. Dawson, J P, Berger, M B, Lin, C C, Schlessinger, J, Lemmon, M A,    and Ferguson, K M (2005). Epidermal growth factor receptor    dimerization and activation require ligand-induced conformational    changes in the dimer interface. Mol Cell Biol 25: 7734-42.-   9. Lange, C, Li, Z, Fang, L, Baum, C, and Fehse, B (2007). CD34    modulates the trafficking behavior of hematopoietic cells in vivo.    Stem Cells Dev 16: 297-304.-   10. Kowolik, C M, Topp, M S, Gonzalez, S, Pfeiffer, T, Olivares, S,    Gonzalez, N, et al. (2006). CD28 costimulation provided through a    CD19-specific chimeric antigen receptor enhances in vivo persistence    and antitumor efficacy of adoptively transferred T cells. Cancer Res    66: 10995-1004.-   11. Szymczak, A L, Workman, C J, Wang, Y, Vignali, K M, Dilioglou,    S, Vanin, E F, et al. (2004). Correction of multi-gene deficiency in    vivo using a single ‘self-cleaving’ 2A peptide-based retroviral    vector. Nat Biotechnol 22: 589-94.-   12. Yam, P, Jensen, M, Akkina, R, Anderson, J, Villacres, M C, Wu,    J, et al. (2006). Ex vivo selection and expansion of cells based on    expression of a mutated inosine monophosphate dehydrogenase 2 after    HIV vector transduction: effects on lymphocytes, monocytes, and    CD34+ stem cells. Mol Ther 14: 236-44.-   13. Pelloquin, F, Lamelin, J P, and Lenoir, G M (1986). Human B    lymphocytes immortalization by Epstein-Barr virus in the presence of    cyclosporin A. In Vitro Cell Dev Biol 22: 689-94.

The invention claimed is:
 1. An in vitro method of enriching orselecting human T-cells comprising a genetically modified EpidermalGrowth Factor Receptor (EGFR) gene, the gene comprising a nucleotidesequence encoding a truncated non-immunogenic endogenous cell surfacemolecule, the cell surface molecule comprising an EGFR Domain III and anEGFR Domain IV; but lacking all of the domains consisting of an EGFRDomain I, an EGFR Domain II, an EGFR Juxtamembrane Domain, and an EGFRTyrosine Kinase Domain; wherein the truncated nonimmunogenic endogenouscell surface molecule (i) does not have endogenous signaling ortrafficking function; (ii) binds a therapeutic anti-EGFR antibody; (iii)does not bind an endogenous EGFR ligand; and (iv) acts as a marker, themethod comprising: contacting a population of cells comprising the humanT cells comprising a genetically modified EGFR gene with an antibodythat binds to the truncated non-immunogenic endogenous cell surfacemolecule.
 2. The in vitro method of claim 1, wherein the geneticallymodified EGFR gene comprises nucleotides 67-1071 of SEQ ID NO:2.
 3. Thein vitro method of claim 1, wherein the genetically modified EGFR geneencodes an amino acid sequence comprising residues 23-357 of SEQ IDNO:3.
 4. The in vitro method of claim 1, wherein the geneticallymodified EGFR gene further comprises a GMCSFR alpha chain signalsequence.
 5. The in vitro method of claim 4, wherein the geneticallymodified EGFR gene comprises SEQ ID NO:2.
 6. The in vitro method ofclaim 4, wherein the genetically modified EGFR gene encodes an aminoacid sequence comprising at least 90% identical to SEQ ID NO:3.
 7. Thein vitro method of claim 4, wherein the genetically modified EGFR geneencodes an amino acid sequence comprising SEQ ID NO:3.
 8. The in vitromethod of claim 1, wherein the genetically modified EGFR gene isinserted into a vector.
 9. The in vitro method of claim 1, wherein thegene is part of a construct which comprises the modified EGFR coupledvia a C-terminal 2A cleavable linker to a chimeric antigen receptorspecific for a tumor associated antigen, wherein the tumor associatedantigen is selected from the group consisting of CD19, CD20 and CD22.10. The in vitro method of claim 1, wherein the modified EGFR is coupledto a codon-optimized anti-CD 19 costimulatory chimeric antigen receptor(CD 19CAR) and a C-terminal 2A cleavable linker.
 11. An in vitro methodof enriching or selecting human T-cells comprising a geneticallymodified Epidermal Growth Factor Receptor (EGFR) gene that is coupled toa CD19CAR and a C-terminal 2A cleavable linker, wherein the gene encodesan amino acid sequence comprising SEQ ID NO:6.
 12. The in vitro methodof claim 11, wherein the genetically modified EGFR gene is inserted intoa vector to transfect the population of human T-cells.
 13. An in vivo ofmethod of depleting T cells comprising a genetically modified EpidermalGrowth Factor Receptor (EGFR) gene, the gene comprising a nucleotidesequence encoding a truncated non-immunogenic endogenous cell surfacemolecule, the cell surface molecule comprising an EGFR Domain III and anEGFR Domain IV; but lacking all of the domains consisting of an EGFRDomain I, an EGFR Domain II, an EGFR Juxtamembrane Domain, and an EGFRTyrosine Kinase Domain; wherein the truncated nonimmunogenic endogenouscell surface molecule (i) does not have endogenous signaling ortrafficking function; (ii) binds a therapeutic anti-EGFR antibody; (iii)does not bind an endogenous EGFR ligand; and (iv) acts as a marker, themethod comprising: administering, to a patient that has been previouslyadministered a population of cells comprising the human T cellscomprising a genetically modified EGFR gene with, an antibody that bindsto the truncated non-immunogenic endogenous cell surface molecule.